Interpretive Summary: Unlike their animal counterparts, plants are non-motile and must adapt to environmental stresses to survive. An increase in calcium within the plant has been associated with many environmental stresses, including salinity. The goal of this research is to understand how plants adapt to salinity stress in order to develop new crops able to grow in high salinity areas. Salinity is composed of two types of stress, an ionic stress and an osmotic stress. Here we demonstrate that tobacco cells exposed to either salt (ionic and osmotic stress) or sorbitol (osmotic stress only), results in a rapid increase in calcium within the cells. Using specific inhibitors that prevent calcium from entering the cell and modulators that prevent the release of calcium from organelles in the cell, we were able to determine that salt induced calcium is derived from both external and internal sources. Neither external calcium influx or calcium release from internal organelles provides adaptation to osmotic stress but the influx of external calcium is responsible for the adaptation of the cells to salt.

Technical Abstract:
The influx of Ca2+ into the cytosol has long been suggested to serve as a signaling intermediate in the acquisition of tolerance to hyperosmotic and/or salinity stresses. Here we use aequorin-transformed suspension-cultured tobacco cells to directly assess the role of cytosolic calcium (Ca2+cyt) signaling in salinity tolerance acquisition. Aequorin luminescence recordings and 45Ca influx measurements using inhibitors of Ca2+ influx (Gd3+ and the Ca2+ selective chelator EGTA), and modulators of organellar Ca2+ release (phospholipase C inhibitors U73122 or neomycin) demonstrate that hyperosmolarity, whether imposed by NaCl or by a non-ionic molecule sorbitol, induces a rapid (returning to baseline levels of Ca2+ within 10 min) and complex Ca2+cyt pulse in tobacco cells, deriving both from Gd3+-sensitive externally derived Ca2+ influx and from U73122- and neomycin-sensitive Ca2+ release from an organelle. To determine whether each of the two components of this brief CA2+ signal regulate adaptation to hyperosmotic shock, the Ca2+ pulse was modified by the addition of Gd3+, U73122, neomycin, or excess Ca2+, and then cells were treated with salt or sorbitol. After 10 min the cell culture medias were diluted with additional hyperosmotic media to reduce the toxic affects of the modulators, and the growth of cells was measured after 1 week. Gd3+ treatment reduced growth in salt relative to control cells but not in sorbitol, and exposure to excess Ca2+ increased growth in salt but not ins orbitol. In contrast, exposure to inhibitors of IP3 formation had no effect on growth in salt or sorbitol. Therefore, although hyperosmotic treatment stimulates both Ca2+ influx and Ca2+ release from an internal Ca2+ depot, only Ca2+ influx has a measurable impact on ionic stress tolerance acquisition in tobacco cell suspensions. In contrast, osmoadaptation in these cells appears to occur independent of Ca2+ signaling.